Crispr transient expression construct (ctec)

Abstract

The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR transient expression construct (CTEC) for a genome editing system.

1 . A CRISPR transient expression construct (CTEC) adapted for ex vivo use and for expression in a host cell of a functional guide-RNA or part thereof that is specific for a target sequence in a target genome, wherein the CRISPR transient expression construct is linear and comprises:
 a guide-RNA expression cassette, and   an additional polynucleotide element, and,   wherein the guide-RNA expression cassette is capable of expressing a functional guide-RNA, or a part thereof, that is specific for a target sequence in a target genome, and wherein the additional polynucleotide element has sequence identity with the target sequence in the target genome.   
     
     
         2 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the functional guide-RNA, or part thereof that is specific for a target sequence in a target genome, is exclusively expressed from the CTEC. 
     
     
         3 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the CTEC is comprised of two or more polynucleotides capable of recombining with each other to yield:
 a guide-RNA expression cassette, and   an additional polynucleotide element,   wherein the guide-RNA expression cassette is capable of expressing a functional guide-RNA, or a part thereof, that is specific for a target sequence in a target genome, wherein the additional polynucleotide element has sequence identity with the target sequence in the target genome.   
     
     
         4 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein in the CTEC, the guide-RNA expression cassette and the additional polynucleotide element are linked by a polynucleotide that comprises a target sequence that corresponds to the guide sequence of the guide-RNA, allowing in vivo cleavage of the guide-RNA expression cassette from the additional polynucleotide element. 
     
     
         5 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the guide-RNA expression cassette is capable of expressing a functional guide-RNA. 
     
     
         6 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the guide-RNA expression cassette comprises a eukaryotic promoter. 
     
     
         7 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the functional guide-RNA, or the part thereof, is encoded by a polynucleotide on the guide-RNA expression cassette and the polynucleotide is operably linked to an RNA polymerase II promoter, to an RNA polymerase III promoter as well as a self-processing ribozyme or to a single-subunit DNA-dependent RNA polymerase promoter, optionally a viral single-subunit DNA-dependent RNA polymerase promoter, optionally a T3, SP6, K11 or T7 RNA polymerase promoter. 
     
     
         8 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the guide-RNA expression cassette is located 3′-of the additional polynucleotide element. 
     
     
         9 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the guide-RNA expression cassette is located 5′-of the additional polynucleotide element. 
     
     
         10 . The CRISPR transient expression construct (CTEC) according to  claim 1 , wherein the CTEC comprises two or more polynucleotide sequences capable of recombining with a vector, optionally a plasmid, to in vivo yield the CTEC integrated into the vector. 
     
     
         11 . A composition comprising the CRISPR transient expression construct (CTEC) as defined in  claim 1 , or a library thereof CRISPR, for expression in a host cell of a functional guide-RNA or part thereof that is specific for one or more target sequence(s) in a target genome. 
     
     
         12 . The CRISPR transient expression construct (CTEC) according to  claim 1  or a composition thereof composition, further comprising a functional polynucleotide-guided genome editing enzyme or an expression construct capable of expressing a functional polynucleotide-guided genome editing enzyme and wherein the functional polynucleotide-guided genome editing enzyme optionally is a Cas9 or a Cpf1. 
     
     
         13 . The construct according to  claim 1 , wherein the host cell is deficient in Non-Homologous End Joining (NHEJ). 
     
     
         14 . A host cell comprising the CRISPR transient expression construct (CTEC) as defined in  claim 1  or comprising a composition comprising said construct. 
     
     
         15 . The host cell according to  claim 14 , further comprising:
 a functional polynucleotide-guided genome editing enzyme, optionally a functional polynucleotide-guided heterologous genome editing enzyme, or   further comprising an expression construct capable of expressing a functional polynucleotide-guided genome editing enzyme, optionally a functional polynucleotide-guided heterologous genome editing enzyme,   wherein the functional polynucleotide-guided genome editing enzyme optionally is a Cas9 or a Cpf1.   
     
     
         16 . The host cell according to  claim 15 , wherein the sequence of the polynucleotide element has been introduced into the genome at the site where the additional polynucleotide element has sequence identity with the sequences flanking the target sequence in the target genome. 
     
     
         17 . The host cell according to  claim 14 , wherein the host cell is deficient in Non-Homologous End Joining (NHEJ). 
     
     
         18 . The host cell according to  claim 14  comprising a polynucleotide encoding a compound of interest. 
     
     
         19 . A method for production of a compound of interest, comprising culturing the host cell according to  claim 18  under conditions conducive to the production of the compound of interest, and, optionally, purifying or isolating the compound of interest.